The immunology, physiology, and genetics shared between humans and swine make swine pathogens an impending threat to human health. Porcine circovirus 2 (PCV2) is a globally distributed non-enveloped immunosuppressive virus demonstrated to infect and lyse human cells in culture. PCV2 infects nearly every tissue in its host and is thus a deterrent to xenotransplantation from swine to humans. Xenotransplantation from swine to human is becoming a practiced solution to the shortage of tissue and organs from human donors. PCV2's ability to rapidly mutate to thrive in its environment was recently demonstrated by emergent strains resistant to commercial PCV2 vaccines. Thus there are a number of reasons why understanding the PCV2 infection mechanism is of urgent priority. The goal of this project is to understand the detailed molecular mechanism by which PCV2 achieves nearly indiscriminate cellular recognition and entry. PCV2 is the smallest autonomously replicating virus, and may therefore define a limit for viruses. The ability of this virus to recognize and enter so many cell types is surprising as non-enveloped animal viruses exhibit a focused range of cell types that they can interact with and enter. This limitation appears to be dependent on the presence of a receptor and/or co-receptor on the cellular surface that the virus capsid can interact with. The project is significant as it provides the opportunity to understand the minimal components necessary to be an autonomously replicating virus, and the capsid determinants that result in broad and nearly indiscriminate cellular recognition and entry. The three independent yet overlapping aims under study are: 1. Define the mechanism by which PCV2 recognizes and interacts with its receptor. Using our crystal structure of PCV2 we hypothesize where the experimentally identified heparan sulfate and chondroitin sulfate B receptors bind. We will test this hypothesis using a combination of biochemical, biophysical and structural biology techniques. 2. Define the mechanism by which PCV2 escapes the endosome. PCV2 has been shown to internalize into different cell types using clathrin-dependent and independent endocytosis. We hypothesize that the arginine rich N-terminus of the PCV2 capsid protein possesses membrane destabilizing activity and used by PCV2 to escape the endosome and enter the cell. Arginine rich polypeptides have been shown to possess such activity. We will use a combination of biochemical and biophysical studies to test this hypothesis. 3. Study the mechanism by which PCV2 enters the cell. The mutants generated to inhibit receptor recognition and endosome escape, in aims 1 and 2 respectively, will be studied for their ability to infect cell types that internalize PCV2 via clathrin-dependent and independent endocytosis. These studies will allow us to reformulate our hypotheses through a cellular lens to describe the mechanism by which PCV2 recognizes and enters so many different cell types. The experiments will also allow us to study whether PCV2 uses a single or multiple mechanisms to enter into different cell types.